Gene deletion and/or replacement is the single most important tool for definitively identifying critical functions of infectivity and virulence in pathogenic bacteria. Yet the tools available to make such gene replacements in pathogenic bacteria have, for the most part, remained unchanged for the last 10 years. While genome sequencing projects continue to increase the number of open reading frames available for genetic analysis, gene knock-out technology in many bacterial systems remains technically cumbersome, and in some cases, unfeasible. This project is designed to explore a novel methodology for the enhancement of gene replacement in pathogenic bacteria. The Red recombination system from bacteriophage lambda, when expressed in Escherichia coli, generates a hyper-recombinogenic phenotype whereby gene replacement occurs at an extremely high efficiency following transformation with small (2-3 kb) linear DNA substrates. This gene replacement scheme is unique in that plasmid-chromosome co-integrants do not have to be formed (or resolved), and prior cloning of the gene of interest is not required. PCR-generated substrates with as little as 40 bp of flanking homology are substrates for efficient Red-mediated gene replacement. The recombination intermediates generated by lambda Red are channeled into the host recombination pathway. It is this "jump start" in the initiation of recombination that likely plays a key role in the generation of the hyper-rec phenotype of lambda Red-containing E. coli. Since most bacteria contain homologs of many of the recombination functions described in E. coli (e.g., recA, recBCD, ruvAB), Red will likely serve to generate the same hyper-rec phenotype when expressed in other (pathogenic) strains of bacteria. This proposal is a test of this hypothesis. This project is designed to generate hyper-recombinogenic strains of Pseudomonas aeruginosa and Mycobacterium tuberculosis by expression of red and phage anti-RecBCD functions in vivo from plasmids, or by replacing the chromosomal recBCD genes with a red-expressing operon. The system can be set up so that the hyper-rec phenotype is transient, resulting in pathogens that are altered only within the gene of interest. This project has the potential to revolutionize the methods of genetic manipulation in microorganisms, leading to faster identification of virulence genes, greater flexibility in the genetic analysis of these genes, and the speedy generation of bacterial mutants for vaccine development.